Moderate intake of total LC ω-3 PUFA (approximately 0.5–1 g/day) was significantly associated with a lower prevalence of depression. Conclusion: In our study, moderate fish and LC ω-3 PUFA intake, but not high intake, was associated with lower odds of depression suggesting a U-shaped relationship.Background: The aim of this analysis was to ascertain the type of relationship between fish and seafood consumption, omega-3 polyunsaturated fatty acids (ω-3 PUFA) intake, and depression prevalence. Methods: Cross-sectional analyses of the PREDIMED-Plus trial. Fish and seafood consumption and ω-3 PUFA intake were assessed through a validated food-frequency questionnaire. Self-reported life-time medical diagnosis of depression or use of antidepressants was considered as outcome. Depressive symptoms were collected by the Beck Depression Inventory-II. Logistic regression models were used to estimate the association between seafood products and ω-3 PUFA consumption and depression. Multiple linear regression models were fitted to assess the association between fish and long-chain (LC) ω-3 PUFA intake and depressive symptoms. Results: Out of 6587 participants, there were 1367 cases of depression. Total seafood consumption was not associated with depression. The odds ratios (ORs) ) for the 2nd, 3rd, and 4th quintiles of consumption of fatty fish were 0.77 (0.63–0.94), 0.71 (0.58–0.87), and 0.78 (0.64–0.96), respectively, and  Unipolar depression is identified as one of the leading causes of burden of disease worldwide, measured in adjusted life years . In addiAccording to the International Society for Nutritional Psychiatry Research, although the growth in scientific research related to nutrition in psychiatry may be recent, it is now at a stage where it can no longer be ignored . In thisThe aims of this study were (1) to cross-sectionally analyze the association between the consumption of different types of seafood products and ω-3 PUFA and depression, (2) to establish the shape of the dose–response curve and the potential existence of a non-linear threshold effect for ω-3 PUFA and, finally, (3) to ascertain if these associations differed by sex, the presence of cardio-metabolic disorders, or several life-style habits in the PREDIMED-Plus trial.http://predimedplus.com/. This study was registered at the International Standard Randomized Controlled Trial  with number 89898870 (registration date: 24 July 2014).This study was based on the cross-sectional analysis of baseline data within the frame of the PREDIMED-Plus trial, a six-year ongoing multicenter, randomized, parallel-group clinical trial conducted in Spain to assess the effect of an intensive weight-loss intervention based on an energy-restricted traditional Mediterranean diet, physical activity promotion, and behavioral support on hard cardiovascular events, in comparison with a control group receiving usual care intervention only with energy-unrestricted Mediterranean diet recommendations. A more detailed description of the PREDIMED-Plus study is available at 2], who met at least three components of the metabolic syndrome (MetS) according to the updated harmonized criteria of the International Diabetes Federation and the American Heart Association and National Heart, Lung, and Blood Institute [A total of 6874 participants were recruited and randomized in 23 recruitment sites from different universities, hospitals, and research institutes of Spain. The eligible participants were community-dwelling adults  with overweight/obesity [body mass index (BMI) ≥ 27 and < 40 kg/mnstitute .n = 259), and participants with missing data in smoking status (n = 28) were excluded from the analyses. Finally, 6587 participants were analyzed.For the present analysis, participants who were outside of predefined limits for baseline total energy intake   acid were collected through six items . Consumption of flaxseed and canola oils was not considered because these oils are not consumed in Spain. Nutrient intakes were computed using Spanish food composition tables ,21. The Depression was collected at baseline and was defined as a self-reported life-time medical diagnosis of depression or the habitual use of antidepressants by the participant. The use of self-reported medical diagnosis of depression collected through a questionnaire has been validated in another Spanish study showing adequate validity . MoreoveInformation about socio-demographic , and lifestyle-related variables  were obtained from the baseline questionnaire.Anthropometric variables were determined by trained staff and in accordance with the PREDIMED-Plus operations protocol. Weight and height were measured with calibrated scales and a wall-mounted stadiometer, respectively. BMI was calculated as the weight in kilograms divided by the height in meters squared.Leisure-time physical activity was assessed using the short form of the Minnesota Leisure Time Physical Activity Questionnaire validated in Spain ,25  proposed by Trichopoulou and was based on the consumption of eight items  . Persona2, continuous), presence of several diseases  at baseline, and total energy intake  and adherence to the Mediterranean Diet . Tests of linear trend across increasing quintiles of exposures were conducted by assigning the medians to each quintile and treating them as continuous variables.Logistic regression models were fitted to assess the relationship between the energy-adjusted consumption of different types of fish and seafood products and intake of ω-3 PUFA (in quintiles) and the prevalence of depression. Odds ratios (ORs) and their 95% CIs were calculated considering the lowest quintile as the reference category. To control for potential confounding factors, the results were adjusted for sex, age, marital status (married/other), educational level , smoking , physical activity during leisure time (quintiles of METs/min-w), BMI  and depressive symptoms assessed through the Beck Depression Inventory-II.p-values for the interaction were calculated with the log-likelihood ratio test.Finally, in order to assess the possible effect modification by sex, type 2 diabetes prevalence, adherence to the MDS, or smoking, product-terms were introduced in the different multivariable models. In addition, We identified 1367 participants at baseline with life-time prevalence of depression. p for trend = 0.759) between intermediate categories of consumption of fatty fish (approximately 10–25 g/day) and the prevalence of depression, suggesting a U-shaped relationship. As compared with the reference category, the ORs (95% CI) for the consecutive quintiles of consumption of fatty fish were 0.77 (0.63–0.94), 0.71 (0.58–0.87), 0.78 (0.64–0.96), and 0.84 (0.69–1.03), respectively. Similarly, moderate consumption of lean fish (approximately 20 g/day) was also associated with lower depression prevalence, with an OR for the 3rd vs. the 1st quintile of 0.77 (0.63–0.94).The association between energy-adjusted quintiles of consumption of different types of seafood products and depression is shown in n = 25) or in which the diagnosis date was very remote . In this sub-sample  the results were no longer significant although the magnitude of effect was quite similar to that observed in the overall sample; the ORs and 95% CI for successive quintiles of fatty fish consumption were: 1 (ref.), 0.85 (0.60–1.20), 0.86 (0.61–1.20), 0.89 (0.63–1.25), and 0.90 (0.64–1.26). In the case of LC ω-3 PUFA, we found the following estimates for the association between quintiles of intake and depression: 1 (ref.), 0.83 (0.60–1.15), 0.83 (0.59–1.15), 0.71 (0.51–1.00), and 0.82 (0.59–1.13).In an ancillary analysis, we excluded from the analyses all depression cases in which age of depression diagnosis was not available . Similarly, none of the product terms was statistically significant in the analysis of LC ω-3 PUFA.In this cross-sectional analysis of the PREDIMED-Plus trial, we observed a non-linear inverse association only for between moderate levels of fish consumption (particularly fatty fish consumption) and LC ω-3 PUFA intake and life-time prevalence of depression and depressive symptoms intensity, but not for the highest levels of intake (U-shaped relationship). The results were not modified by sex, by the presence of type 2 diabetes, or by several life-style factors such as smoking.Although a recent study has found a dose–response inverse relationship between fish consumption and depression with an OR of 0.52 (95% CI: 0.37–0.74) for those participants with the highest consumption of fish (≥4 times/week) as compared to those with a consumption lower than 1 time/week , a meta-Several possible explanations may contribute to a better understanding of this non-linear association between fish consumption and depression. One of the more feasible explanations is the intake of other nutrients that could counteract the effect of fish or LC ω-3 PUFA intake on depression, including ω-6 PUFA intake as some authors have suggested ,31. ActuThe results observed for specifically fatty and lean fish were not reproduced for other kinds of seafood products or for canned fish. The content in LC ω-3 PUFA could differ regarding fish/seafood sources, being higher in fatty fish and lower in other sea products such as mollusks, crayfish, or octopus. Although the level of LC ω-3 PUFA in canned fish (mainly tuna and anchovy in Spain) seems to be similar to that found in fresh fish , the conThe role of LC ω-3 PUFA in depression has been extensively evaluated as coadjuvant in antidepressant treatment ,9. The rA large number of studies have reported the possible role of inflammation in depression through mechanisms such as activation of the hypothalamic–pituitary–adrenal axis, tryptophan depletion, neurotransmitter transport and metabolism disturbances, and decrease in brain-derived neurotrophic factor availability ,36. All The strengths of this study are its large sample size, the exposed populations consisting of people of both sexes, the adjustment for a wide array of potential confounders, and the use of validated tools to assess information. Some potential limitations of our study also need to be mentioned. The cross-sectional design of the study does not afford us the possibility of establishing any causal association between fish consumption and depression, and the presence of a possible reverse causality bias cannot be excluded. In fact, the presence of a depressive disorder could lead to less healthy dietary habits including lower fish consumption. Contrarily, participants with depression could also increase their fish or ω-3 PUFA intake to improve their depressive condition. Another possible caveat might be that our participants are not representative of the general Spanish population. Our participants were aged between 55 and 75 years, were overweight or obese, and all met criteria for metabolic syndrome. Nevertheless, the lack of representativeness does not preclude the establishment of associations . Self-reIn conclusion, the findings from the current study support the idea that moderate intake of fish and LC ω-3 PUFA (U-shaped relationship) may protect against depression independently of the sex differences, the presence of cardiometabolic disturbances, or life-style habits. More studies with longitudinal design are needed to confirm the reported results and definitively establish the role of seafood products and ω-3 PUFA in depression development.


L-arginine oxidation, is an essential trigger for signaling processes involved in structural and metabolic changes in muscle fibers. Recently, it was shown that L-arginine administration prevented the decrease in levels of the muscle cytoskeletal proteins, desmin and dystrophin, in rat soleus muscle after 14 days of hindlimb unloading. Therefore, in this study, we investigated the effect of L-arginine administration on the degree of atrophy changes in the rat soleus muscles under unloading conditions, and on the content, gene expression, and phosphorylation level of titin, the giant protein of striated muscles, able to form a third type of myofilaments—elastic filaments. A 7-day gravitational unloading [hindlimb suspension (HS) group] resulted in a decrease in the soleus weight:body weight ratio , indicating muscle atrophy development. The content of intact titin (T1) decreased  and the content of proteolytic fragments of titin (T2) increased  in the soleus muscle of HS rats, compared to control rats. The titin gene expression and phosphorylation level of titin between these two groups were not significantly different. L-Arginine administration under 7-day gravitational unloading decreased the degree of atrophy changes and also prevented the decrease in levels of T1 in the soleus muscle as compared to HS group. Furthermore, L-arginine administration under unloading resulted in increased titin mRNA level  and decreased phosphorylation level of T2 , compared to those in the HS group. These results suggest that administration of L-arginine, the NO precursor, under unloading decreased the degree of atrophy changes, increased gene expression of titin and prevented the decrease in levels of T1 in the rat soleus muscle. The results can be used to search for approaches to reduce the development of negative changes caused by gravitational unloading in the muscle.Nitric oxide (NO), produced by NO-synthases via  L-arginine. NO-synthase actively participates in the regulation of protein and energy metabolism in skeletal muscles, including the regulation of protein synthesis and degradation (Nitric oxide (NO) is an essential trigger for signaling processes that lead to structural and metabolic changes in muscle fibers . It is pradation .2+ via Ca2+-dependent binding of the enzyme to the calcium-calmodulin complex , associated with dystrophin in the subsarcolemmal region, is mostly expressed in skeletal muscles . Neurona complex . Neuronaed to NO .Muscle NO concentration has been shown to increase with an increase in muscle contractile activity  and decrin vitro via S-nitrosylation of the active site cysteine  degree of atrophy changes in rat soleus muscles after hindlimb unloading, (ii) level and gene expression of titin, and (iii) changes in levels of titin phosphorylation in rat soleus during unloading. New data were obtained showing that administration of L-arginine, the NO precursor, under gravitational unloading decreased the degree of atrophy changes, increased gene expression of titin and prevented the decrease in levels of T1 in the rat soleus muscle.Changes in titin gene expression were observed in mice skeletal muscles that were atrophied after a 30-day-long space flight . Increasnloading . Howeverad libitum. This study was carried out in accordance with the recommendations of –1). The depth of anesthesia was evaluated by testing the pedal withdrawal reflex (toe and foot pad pinch).Fifteen 3-month-old male Wistar rats (190 ± 5 g) were obtained from the certified Nursery for laboratory animals, Institute of Bioorganic Chemistry of the Russian Academy of Sciences . The animals were housed in a temperature-controlled room under a 12:12 h light–dark cycle; food pellets and water were provided n = 5/group): (1) C, cage control; (2) HS, 7-day hindlimb suspension; and (3) HSL, 7-day hindlimb suspension + L-arginine. 7 days were chosen because preliminary studies demonstrated the development of atrophy in human soleus fibers and significant decrease in the titin content after 7 days gravitational unloading  via intramuscular injections. The dose of L-arginine was previously tested and found to be optimal (The hindlimbs were unloaded using a standard rodent hindlimb suspension (HS)/unloading model . Brieflynloading . Animals optimal . The ratTM Total RNA Fatty and Fibrous Tissue kit, according to the manufacturer’s protocol . The total RNA quality was assessed by visualizing the integrity of the 18S and 28S rRNA after electrophoresis in 1% agarose gels. Total RNA concentration was determined via spectrophotometry . RNA solutions were stored at −75°C.Total RNA was isolated from the samples using the Aurum15 primers . Primers specific for gene fragments of titin and GAPDH were selected using Vector NTI software. The following primer pairs were used: titin (N2A isoform) 5′-CAGCAGCCAAGAAAGCCGCT-3′ (forward), 5′-CACCACTCTGATACTCTGAGGCTCTG-3′ (reverse) and GAPDH gene 5′-GCAAGAGAGAGGCCCTCAG-3′ (forward), 5′-TGTGAGGGAGATGCTCAGTG-3′ (reverse). Nucleotide gene sequences were obtained from the NCBI GenBank database. Real-time PCR was performed using a DT-322 amplifier , Taq-DNA polymerase (Evrogen), and SYBR Green I fluorescent dye . PCR was performed as follows: (i) hot start at 95°C for 5 min, (ii) denaturation at 95°C for 15 s, (iii) primer annealing at 64°C for 20 s, and (iv) extension at 72°C for 20 s. Stages 2 to 4 were repeated 35 times. The amplification products were analyzed by electrophoresis in a 7% polyacrylamide gel. The PCR products were isolated from the gel according to the Cleanup Standard protocol . DNA fragments were sequenced in Evrogen. BLAST program software was used for identification of the PCR product  and oligo-dTduct see . The chat method , and theChanges in titin-1  and T2 (proteolytic fragments) content were determined using polyacrylamide slab gels . The gels were strengthened with agarose according to the Tatsumi–Hattori technique  with somTM Touch Imaging System (Bio-Rad). The data were processed using the Image Lab Software (Bio-Rad). Finally, the gels were stained with Coomassie brilliant blue G-250 and R-250, mixed in a 1:1 ratio, to determine the total protein content.The level of titin phosphorylation was determined using a previously described method  with minp < 0.05.Gels were digitized, and the data were processed using the TotalLab v1.11 software . To determine the titin content relative to that of the myosin heavy chain, the total optical density of the myosin heavy chain peak and the total optical density of the T1 and T2 peak were determined on the same gel, as described previously . It is kp < 0.05) and lower soleus weight:body weight ratio  was observed in the HS rats, compared to the C rats (p = 0.073), and tendency to reduction in the soleus muscle weight:body weight ratio   .p < 0.05), and a 66.7% increase in T2 content (p < 0.05) in the soleus muscle (p = 0.086) in the soleus muscles.Compared to the C group, the HS group showed a 22.4% decrease in T1 content (s muscle . The difs muscle . Comparep = 0.333) in the soleus muscles (p = 0.234) and 28% (p < 0.05) lower than that in the C and HS groups, respectively, Compared to the C group, the HS group showed a tendency to higher T2 phosphorylation levels , compared to that in rats from the C group. Compared to the HS group, L-arginine administration under 7-day gravitational unloading showed a 76% increase in the titin mRNA level  , and caliscussed . Moreovenloading . In our nloading .Changes in titin gene expression and their possible role in alterations of the content of this protein under unloading remains unclear. Titin gene expression was shown to be increased (by 70%) in the atrophied gastrocnemius muscle of mice after a 30-day-long space flight . HoweverL-arginine administration in our work, suggesting the enhanced synthesis of this protein. This increase titin gene expression may be regulated via NO signaling. In 2008, it was shown that brain-derived neurotrophic factor triggers NO synthesis and S-nitrosylation of histone deacetylase 2, subsequently leading to gene activation in neurons (However, increased titin gene expression in the rat soleus muscle was observed after unloading with  neurons . A later neurons . NO-depe neurons  or GSK3β neurons . Our datIn vivo titin phosphorylation is well known (1,2. Phosphorylation is known to modify the stiffness of titin molecules (ll known  and the ll known . Many poll known 1,2. Phoolecules . In receolecules . The rololecules . For insolecules . We founolecules . Accordiolecules . It has olecules .L-arginine during unloading caused hypophosphorylation of titin in the rat soleus muscle, that accompanied by a smaller decrease in the level of T1. Collectively, these results suggest that hypophosphorylation of titin, especially T2-part, decreases the susceptibility of this protein to proteolysis. This may be indirectly supported by finding that GSK3β inhibition in mice prevented the phosphorylation and depolymerization of desmin and blocked atrophy upon fasting or denervation. Mass spectrometry analysis identified GSK3-β and calpain-1 bound to desmin and catalyzing its disassembly (In the present study, we showed a tendency to higher T2 phosphorylation level after HS. This tendency was accompanied by the enhanced proteolytic degradation of T1. These findings are in agreement with the assumption mentioned above. Administration of assembly . This stL-arginine, the NO precursor, under gravitational unloading decreased the degree of atrophy changes, increased gene expression of titin and prevented the decrease in levels of T1 in rat soleus muscle. The results can be used to search for approaches to reduce the development of negative changes caused by gravitational unloading in the muscle. Because of limited n-number and high data variability, further research is required to provide evidence of changes in the phosphorylation level of titin and gain insight into how the post-translational modification affect the susceptibility of titin to proteolysis. To find answers to these research questions is the aim of our future studies.In conclusion, we showed that administration of This study was carried out in accordance with the recommendations of All datasets generated/analyzed for this study are included in the manuscript/TN, BS, and IV conceived and designed the work. AU, YG, NS, YL, and SB performed the experiments. AU, BS, and IV analyzed the data and wrote the manuscript. AU and YG prepared the figures. AU, BS, and IV reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The application of quinoline-based compounds for the treatment of malaria infections is hampered by drug resistance. Drug resistance has led to the combination of quinolines with other classes of antimalarials resulting in enhanced therapeutic outcomes. However, the combination of antimalarials is limited by drug-drug interactions. In order to overcome the aforementioned factors, several researchers have reported hybrid compounds prepared by reacting quinoline-based compounds with other compounds via selected functionalities. This review will focus on the currently reported quinoline-based hybrid compounds and their preclinical studies. Anopheles mosquito when it takes a blood meal. Pregnant women and young children in the sub-tropical regions are more prone to malaria infection. In 2015, 214 million infections were reported, with approximately 438,000 deaths and the African region accounted for 90% of the deaths  pyrimidine was 56 fold less than that of chloroquine, with an IC50 of 3.6 nM against a chloroquine-resistant strain sulfonyl}-N-acetylaniline) was sevenfold less active compared to quinine -N-methylpropane-1,3-diamine (51) from 4-aminoquinoline and imipramine -thiazin-4-one. The compounds formed a complex with hematin, thereby inhibiting the formation of β-hematin [P. falciparum (30-fold). The compound in which the terminal amine side chain is replaced with a less bulky methyl group was as potent as chloroquine against the D2 clone and about 7-fold more potent against the W2 clone [50 values of 26 and 19 nM and against multi-drug-resistant strain W2 with IC50 values of 49 and 42 nM, respectively [Plasmodium falciparum with an IC50 of 5.23 ng/mL and 7.88 ng/mL. The compound with an oxalamide moiety inhibited 70.45% on day 4 against chloroquine-resistant strain N-67 of Plasmodium yoelii in vivo [Plasmodium falciparum with an IC50 of 0.096 μM [50 values ranged between 1.39 and 1.69 mg/mL for the most potent compounds. They were effective against P. berghei in mice in vivo. However, the (pyrazol-1-yl)chloroquine compounds were inactive, indicating that the hydroxyl functionality in the pyrazole ring was important for antimalarial activity [Hybrid compounds with a dibenzylamino moiety were effective against chloroquine-resistant and -sensitive strains of nificant . Ribeironificant . Some ofnd 23 nM . The act102.4 nM . Solomon-hematin . Khan et-hematin . The comW2 clone . Cornut ectively . Sunduruectively . Compoun in vivo . The mos0.096 μM . Compoun0.096 μM . Cunico 0.096 μM . The IC5activity .P greater than 5 and if the sum of nitrogen and oxygen atoms is greater than 10 [P. falciparum (D6 and W2) with IC50 values 0.038–0.044 μM which was equipotent to artemisinin, a potent antimalarial drug with IC50 = 0.045 μM [P. falciparum had log P values nearly equal to quinine and chloroquine [H-pyrrol-2,5-dione moiety. The drug-likeness score was in a range of 0.16 to 0.87 based on Lipinski’s rule of five and comparable to chloroquine with a score of 0.67 [Although hybrid compounds are potential molecules which can overcome drug resistance, some of them have a large size which can limit their oral bioavailability. Lipinski’s rule, also known as the “rule of five”, was proposed for drug-likeness, which states that poor permeability of a compound will occur if the hydrogen-bond donors are greater than five, if the molecular mass is greater than 500 with calculated log  than 10 . However than 10 ,92. The  than 10 . It is a than 10 . There a than 10 . Compoun than 10 . Sharma  than 10 . Compoun than 10 . Maurya oroquine . Rojas e of 0.67 . Kumar e of 0.67 . On the Plasmodium parasites. Their ability to overcome resistance is attributed to their lipophilicity. However, hybrids with rigid linkers were not active biologically when compared to hybrids with flexible linkers. Quinoline-trioxolanes inhibit heme aggregation within the parasite, either by forming unpolymerizable heme drug adducts or by stacking the quinoline fragment with heme. The nature of the linker has an influence on the antimalarial activity of the compounds in vitro and in vivo. Hybrid compounds containing a quinoline moiety and antibiotics or anticancer drug with good antimalarial activity were also reported. They were effective against blood and liver stage malaria with high percentages of inhibition of parasite multiplication. The design of the compounds influenced the antimalarial activity. Selected 4-aminoquinoline-pyrimidine hybrids are potent antimalarials and some of them were 6–26 times more effective compared to chloroquine. The mode of action of the compounds is by heme-detoxification. The length of the chain linker did not have effect on the antimalarial activity of some of the compounds, however, they were selective towards either chloroquine-sensitive strain or chloroquine resistant strain depending on the nature of the linker. Hybrid compounds with piperazinyl linker nullified the antiplasmodial activity which is attributed to the steric restrictions hindering a face-to-face stacking of the quinoline unit with heme and ineffective inhibition of the heme polymerization to hemozoin. Quinoline-sulfonamide hybrids exhibit good antimalarial activity but most of them were less active when compared to chloroquine. They acted by inhibiting parasite growth without interfering with the integrity of the red blood cell membrane. To date, the structural modifications in heterocyclic systems have provided advancements in the development of drugs for the treatment of malaria, however, some heterocyclic systems have not been explored, which could provide additional new pharmacophores and drugs for the treatment of malaria. There is no doubt that the continuous advancement in the development of quinoline heterocyclic compounds will give rise to potent antimalarials to overcome drug resistance which is common with the currently used antimalarials.Hybrid compounds with antimalarial activity containing quinoline are cost effective, with reduced risk of drug-drug interactions. In the design of the hybrid compounds, the linker influences the pharmacokinetic behaviour of the drugs. There are several reports of hybrid compounds containing quinoline with good antimalarial activity. Artemisinin-quinoline hybrid compounds exhibited good antimalarial activity when compared to the individual drugs. However, some of the hybrid compounds were found to be less effective compared to the individual drugs. The chain length between the artemisinin and quinoline moieties influenced the antimalarial activity of the compounds. Quinoline-ferrocene analogues act as resistance-reversing agents, thereby inhibiting resistance of the


Under optimal conditions, the present immunoassay measures CRP in a linear range from 0.4–200 nM (0.047–23.6 µg mL−1), with a detection limit of 0.15 nM  and sensitivity of 90.7 nA nM−1, in addition to a good reproducibility and storage stability. The analytical applicability of the presented immunoassay is verified by CRP measurements in human blood serum samples. This work provides the basis for a low-priced, safe, and easy-to-use point-of-care immunosensor assay to measure CRP at clinically relevant concentrations.C-reactive protein (CRP) is one of the most expressed proteins in blood during acute phase inflammation, and its minute level increase has also been recognized for the clinical diagnosis of cardio vascular diseases. Unfortunately, the available commercial immunoassays are labour intensive, require large sample volumes, and have practical limitations, such as low stability and high production costs. Hence, we have developed a simple, cost effective, and label-free electrochemical immunoassay for the measurement of CRP in a drop of serum sample using an immunosensor strip made up of a screen printed carbon electrode (SPE) modified with anti-CRP functionalized gold nanoparticles (AuNPs). The measurement relies on the decrease of the oxidation current of the redox indicator Fe Milli-Q water and 0.1 M PBS (pH 7.2) were used for reagent preparation and dilution. The PBS was deaerated prior to use.Human blood serum , human CRP, anti-human CRP developed in goat, bovine serum albumin (BSA), potassium ferrocyanide KThe blood serum was diluted 10 times in PBS. A 30 µL of the sample (or CRP standard solution) was placed onto the imunosensor strips and the concentration of CRP was measured using amperometric technique. After an incubation time of 30 min, the electrodes were washed with PBS and a drop of 50 µL of 0.5 mM ferrocyanide that was prepared in PBS was dropped onto the electrodes. Finally, a standard addition calibration curve was used to quantify the concentration of CRP. Throughout, the dilution factor was taken into account. 2. Once the preparation was accomplished, the immunosensor strip was equilibrated in PBS cycling the voltammetric potential between −0.6 V and +0.6 V until the current stabilised. A scanning electron microscope (SEM)  with an electron energy adjusted to 2 keV was used to obtain the morphological images of the samples. The three electrode electrochemical workstation CHI 1240B  was used for cyclic voltammetric and amperometric studies. Screen printed electrode  comprising of carbon counter electrode, an Ag/AgCl reference electrode, and anti-CRP functionalized AuNPs modified working electrode were used as immunosensor strip. The working electrode surface area was 0.071 cm2SO4 solution. A potential ranging from −0.5 to +1.0 V was swept for 10 cycles at a scan rate of 50 mV s−1. Such a treatment removes the organic ink constituents/contaminants and increases the surface functionalities. Then, the AuNPs were electrodeposited by placing the mixture of 0.5 mM HAuCl4·3H2O and 0.1 M KNO3 solution onto the SPE and sweeping the potential from +0.9 to 0 V for five cycles at a rate of 50 mV s−1. To functionalize the AuNPs with anti-CRP, a self-assembled monolayer (SAM) of cysteine with a carboxylic acid (-COOH) functional head group was anchored by its AuNPs-thiol bond. Subsequently, 1 mM L-cysteine solution was poured onto the AuNPs-SPE and kept at room temperature for 4 h. After the SAM was formed, the surface of the working electrode was washed with ethanol and water before drying with a flow of nitrogen gas. In order to ensure a stable terminal activation of -COOH groups, the SAM was treated with coupling reagents containing 0.4 M EDC and 0.1 M NHS for 30 min prior to gentle rinsing cycles using PBS twice to remove the poorly attached linker molecules. Subsequently, the prepared surface was incubated with a 10 µM anti-CRP solution at an ambient temperature for 1 h. The reaction of the primary amine (-NH2) groups of the antibodies with the -COOH groups on the AuNPs resulted in a formation of stable amide bonds, as schematically shown in Prior to modifying the immunosensor, the SPE was pre-treated in 1 M HFor evaluating the immunoreaction between CRP and anti-CRP, a known concentration of CRP that was prepared with PBS solution was placed over the sensing element and was allowed to react with anti-CRP for 30 min. To remove the non-specifically attached CRP, the electrode was thoroughly washed with PBS and dried before every electrochemical experiment.The morphological images of the bare SPE and AuNPs modified SPE are shown in 64− at a scan rate of 50 mV s−1. Typical electrochemical responses are shown in The electrochemical characteristics of the AuNPs-SPE, anti-CRP-SAM-AuNPs-SPE, BSA-anti-CRP-SAM-AuNPs-SPE, and CRP-anti-CRP-SAM-AuNPs-SPE (1 nM CRP) were investigated using cyclic voltammetry in PBS containing 0.5 mM Fe(CN)C and D correspond to the concentration and diffusion coefficient of Fe(CN)64−, respectively. The parameters pI, n, γ, and A are attributed to the maximum current response, the number of transferred electrons per molecule, the scan rate, and the effective surface area. The calculated surface coverage of anti-CRP on SAM-AuNPs-SPE was 22.6 nmol cm−2, which is about 13 times higher than for the bare SPE (1.7 nmol cm−2), suggesting that the enhanced immobilization of anti-CRP is related to the larger surface area and the higher electron transfer rate that is offered by the spherically shaped AuNPs (20–60 nm) [To evidence the effect of AuNPs, bare SPE, SAM-AuNPs-SPE, anti-CRP-SAM-AuNPs-SPE, and anti-CRP-SPE were incubated in 1 nM CRP and their electrochemical responses were recorded by applying the same conditions as described above. The corresponding responses are shown in 0–60 nm) .−1 were recorded, as displayed in The optimized gold salt concentration (0.5 mM) and electrodeposition time  were used from our earlier report  to achieThe performance of the immunosensor is mainly influenced by the antibody concentration, pH, and CRP incubation time, and, hence, these parameters have been carefully optimized. The electrochemical response of the SAM-AuNPs-SPE decreases while increasing the anti-CRP concentration from 200 nM to 10 μM and stabilizes at higher concentration, as shown in The effect of incubation time on the amperometric current response was furthermore investigated. During incubation, the CRP reaches the anti-CRP on the SAM-AuNPs-SPE surface and takes some time to form an immunocomplex. With an increasing incubation time, the current response decreases and stabilizes after 30 min, as shown in 64−. The observed oxidation currents, measured after a time lapse of 500 s, vs. the CRP concentration are plotted in −1) with a detection limit of 0.15 nM  and a sensitivity of 90.7 nA nM−1. The electroanalytical characteristics  of the immunosensor are similar to earlier that reported label-free methods as shown in Under optimized conditions, the determination of CRP concentration was performed using amperometry technique at constant oxidation potential +0.3 V. The electrochemical response of the anti-CRP-SAM-AuNPs-SPE was considered as base line (control) and the corresponding variations with respect to the concentration of the CRP were measured. Since the determination of CRP concentration is based on the selective immunoreaction between CRP and anti-CRP, the present immunosensor is highly selective . HoweverThe stability of the immunosensor was tested when stored at 4 °C. The electrochemical current responses decreased by 3.6% after two weeks, and 6.8% after four weeks D, indicaThere are several amperometric sandwich immunoassays that are reported for point-of-care CRP analysis ,30,36,37In summary, a simple label-free electrochemical immunoassay has been successfully tested for the measurement of the important cardiac biomarker CRP using SPE modified with anti-CRP functionalized AuNPs. The well characterized and optimized immunosensor strip shows good analytical performances, such as wide linear detection range, low detection limit, and high sensitivity, in addition to excellent reproducibility and stability. The present immunoassay enabled for measuring CRP concentrations in human blood serum samples, and is, hence, highly suited for quality health care management. It could be adopted for point-of-care analysis. Furthermore, the presented configuration could inspire researchers to develop further label-free electrochemical immunosensors for the measurement of different clinical biomarkers.


Small nucleolar RNAs (snoRNAs) are a different type of small non-coding RNAs that can also be reliably measured in plasma, but have only been studied sporadically. In this study, we aimed to identify RNA-biomarkers that can distinguish between different exercise regimes and that entail clues about muscle repair and recovery after prolonged exhaustive endurance exercise. We compared plasma microRNA profiles between two cohorts of elite cyclists, subjected to two different types of exercise regimes, as well as a cohort of patients with peripheral artery disease (PAD) that were scheduled to undergo lower limb amputation, due to critical limb ischemia. In elite athletes, muscle tissue recovers quickly even after exhaustive exercise, whereas in PAD patients, recovery is completely impaired. Furthermore, we measured levels of a specific group of snoRNAs in the plasma of both elite cyclists and PAD patients. Using a multiplex qPCR screening, we detected a total of 179 microRNAs overall, of which, on average, 161 microRNAs were detected per sample. However, only 30 microRNAs were consistently expressed in all samples. Of these, two microRNAs, miR-29a-3p and miR193a-5p, that responded differently two different types of exercise, namely exhaustive exercise and non-exhaustive endurance exercise. Using individual rt/qPCR, we also identified a snoRNA, SNORD114.1, which was significantly upregulated in plasma in response to endurance exercise. Furthermore, two microRNAs, miR-29a-3p and miR-495-3p, were significantly differentially expressed in athletes compared to PAD patients, but only following exercise. We suggest that these two microRNAs could function as markers of impaired muscle repair and recovery. In conclusion, microRNAs miR-29a-3p and miR-193a-5p may help us distinguish between repeated exhaustive and non-exhaustive endurance exercise. MicroRNA miR-29a-3p, as well as miR-495-3p, may further mark impaired muscle recovery in patients with severe critical limb ischemia. Furthermore, we showed for the first time that a circulating snoRNA, SNORD114.1, is regulated in response to exercise and may be used as biomarker.Circulating microRNAs have proven to be reliable biomarkers, due to their high stability, both  Regular exercise at moderate intensity has clear benefits for general health, overall fitness, quality of life and importantly, cardiovascular physiology. Exercise at elite level however, is far more demanding of the body and muscles. During high intensity endurance exercise, muscle glycogen is the major energy substrate and metabolic stress develops. Moreover, exercise triggers an increase in cytokine production, attraction of leukocytes, upregulation of pro-angiogenic stimuli and an overall pro-inflammatory state . In healIn patients with peripheral artery disease (PAD), poor circulation leads to a chronic ischemia and low levels of nutrients in the affected limb(s). PAD is caused by chronic accumulation of atherosclerotic lesions in the arteries of the limbs, generally of the leg . BesidesBoth in exercise physiology and in PAD pathology, researchers have been trying to identify reliable biomarkers to follow muscle repair and recovery. In exercise physiology, such biomarkers could be used to improve training and recovery schedules to optimize competitive performance. Whereas in PAD, such biomarkers may assist vascular surgeons and PAD patients in their joint decision-making before surgical intervention, it being revascularization or even amputation. Although the respective perspectives are very different, potentially, both fields of research could stand to benefit from each other.In recent years, circulating microRNAs have proven to be highly reliable biomarkers . MicroRNO-ribose-methylation  has increased as well over the past few years, but little is known about their role in exercise and muscle physiology. Similar to microRNAs, snoRNAs are small, highly conserved RNAs. Their canonical function is to stabilize other RNA species, either through 2′hylation , or throhylation .in vivo, small non-coding RNAs have also proven to be highly stable ex vivo, resisting degradation due to poor sample handling, repetitive freeze-thawing and long-term storage. This remarkable stability may give small non-coding RNAs a further advantage as biomarkers  were recruited for our cohort. All subjects had competitive experience at national or international level and trained, 16 ± 1 h weekly, of which 12 ± 2 h was cycling.This study was carried out in accordance with the recommendations of Guidelines for Human Exercise Trials, Norwegian School of Sport Sciences’ Ethics Committee. The protocol was reviewed by Regional Ethic Committee of Norway (2011/1298) with the decision that the research project was outside the Act on Medical Health Research, confirmed in letter of exemption . The study was conducted according to the Declaration of Helsinki and participants gave their informed written consent after oral and written information about the study.2max (W73%).Before trial start, subjects completed a familiarization session with a self-paced warm up and a possibility for subjects to adjust the ergometer  geometry to individual preferences, as previously described . Linear 73%, followed by another 5 min of recovery. Subsequent intervals of 20 min at W73% with 5 min of rest between intervals were performed until subjects were unable to maintain predetermined cadence ± 5RPM. After exhaustion, athletes were given 5 min of recovery, followed by repeated intervals of 60 s at 90% VO2max-sprints with 60 s of rest between sprints until voluntary exhaustion. Blood samples were drawn at baseline and at 120 min after the exercise session.On the first trial day an exhaustive exercise session was performed. The session started with a warm up as described previously . After 573%. After 5 min rest, a time test was performed with a predetermined amount of work [Work (kJ) = Power at VO2max (W) ∗ 1800 s]. Initial workload was set to W73%, but the athletes were free to adjust workload to personal preference. Five minutes after the time-trial participants performed a 10 s all-out effort sprint. Blood was drawn before warm up and immediately after completion of the exercise session.On the second day, roughly 18 h after finishing the first exhaustive exercise session, subjects completed the same warm up as on the previous day. After warm up, 5 min of recovery was followed by a 10 s sprint, followed by 30 min cycling at Wg, 10 min) at 4°C. Plasma was transferred to 1.5 ml tubes and stored at -80°C until analysis.Blood was drawn from the antecubital vein using a Teflon catheter  into EDTA-coated tubes and centrifuged  from the Leiden University Medical Center biobank Ampubase were included in our study. All patients were scheduled for lower limb amputation, either below or above the knee joint as a consequence of critical limb ischemia. Inclusion criteria for the biobank were a minimum age of 18 years and lower limb amputation, excluding ankle, foot, or toe amputations. Exclusion criteria were confirmed or suspected malignancy and inability to give informed consent.The study was approved by the Medical Ethics Committee of the Leiden University Medical Center (Protocol No. P12.265) and written informed consent was obtained from all participants.g at 4°C. Plasma was then aliquoted and the plasma closest to the buffy coat was discarded. Aliquots were stored at -80°C until analysis.Blood samples were collected pre-operatively. Sample collection followed standard hospital procedures. Blood was collected in citrate coated tubes and immediately put on ice. Samples were processed within 30 min of collection and were centrifuged for 10 min at 2800 Thirteen highly trained, male endurance cyclists  were recruited for this study. All subjects had competitive experience at national or international level and trained for 11.5 ± 1.2 h weekly, of which 8.8 ± 1.4 h was cycling. Due to sample depletion, only 10 athletes with complete sets of samples were included in our analyses.All subjects were informed of any potential risks before acquiring their written informed consent. The project was approved by Regional Ethics Committee (2013/1528) of Norway, and performed according to the Declaration of Helsinki.The initial pre-trial testing, analysis and equipment used in the second cohort was identical to the EEE cohort, described above.2max, respectively. The exercise session was started with a 10 s all-out sprint immediately followed by 4 min of active (100W) or passive restitution. After initial restitution, participants cycled 30 min at W73%, followed by a 60 min time trial separated by 5 min of restitution. The session ended with another 10 se all-out sprint.On the day of the trial, subjects started with a standardized warm up of 5–2 min of 50, 55, 60, and 75% of the pre-trial VOBlood samples were drawn before exercise, immediately after exercise completion as well as after 2 and 5 h of restitution.g) at 4°C, after which plasma was aliquoted and stored at -80°C until subsequent analysis.Blood was drawn from the antecubital vein using a Teflon catheter  into EDTA-coated tubes. Samples were stored on ice until centrifugation  for RNA isolation, reverse transcription and Multiplex Circulating MicroRNA qPCR Panel for microRNA expression analysis, using their in-house products and protocols. Samples were selected on relative homogeneity in age and other physical parameters.Total RNA from 100 μL of plasma was extracted using the miRCURY RNA Isolation Kit – Biofluids , including rDNase treatment to prevent DNA contamination. RNA was stored until further analysis at -80°C.RNA was reverse transcribed using the miRCURY LNA Universal microRNA PCR, Polyadenylation and cDNA Synthesis Kit. Each microRNA was assayed by qPCR using microRNA Ready-to-Use PCR, Serum/Plasma Focus panel using ExiLENT SYBR Green Master Mix. Negative controls were included as master mix without cDNA template. Amplification was performed using the LightCycler 480 Real-Time PCR System . Amplification curves were analyzed using the Roche LC software, both for determination of Cq and for melt curve analysis.Amplification efficiency was calculated using Exiqon’s in-house algorithms similar to the LinReg software. Melting curves and temperatures of all assays were within specifications of the assays. All measurements with five Cqs less than the negative control and with a Cq < 37 were included in the analysis. Data that did not meet these criteria were omitted from further analyses. All data were normalized to the average of all assays in all samples (average Cq – assay Cq).For snoRNA and confirmatory microRNA analyses, total RNA was isolated from all available plasma samples using TRIzol-LS Reagent, according to the Manufacturer’s protocol .v= Σ/μ) was calculated and triplicates resulting in a Cv> 0.02 were excluded. SnoRNA expression was normalized to U6.For snoRNAs analyses, RNA was reverse transcribed using ‘High-capacity RNA to cDNA kit’  and quantified by RT-qPCR using SybrGreen reagent  on the VIIa7 . All qPCR reactions were performed in technical triplicates. The Coefficient of Variance . All qPCR reactions were performed in technical triplicates. The Cp-Values of < 0.05 were regarded as significant, p-values < 0.1 were regarded as a trend. Normalized relative expression values are presented as mean ± SEM. Due to large inter-sample variation, Grub’s test was used to identify significant (α < 0.05) outliers in ncRNA measurements.All statistical analyses, unless otherwise specified, were performed using GraphPad Prism 7 . post hoc test for multiple comparisons.One-way analyses of variance (ANOVA) was used to determine differences in expression levels over time within a cohort. To identify significant effects within ANOVAs, the Tukey’s Honest Significant Difference-test was used as a t-test were used, paired or unpaired, depending on within or between group comparisons. Values are presented as the fold change between groups.When comparing mean expression between different cohorts two-tailed Student’s Figure 2).Samples drawn from five participants of the EEE and five participants of the PAD cohorts were sent to Exiqon in Denmark for multiplex rt/qPCR expression profiling. The multiplex panel measures a total of 179 microRNA, of which, on average, 161 microRNAs were detected per sample. 30 microRNAs were consistently detected in all samples. Although all samples passed quality control, the microRNA content, both in number of detected microRNAs and expression levels of the detected microRNAs, was lowest in PAD cohort samples  that all PAD samples cluster together and are clearly different from the athletes’ samples. When looking at the athletes’ samples, overall clusters are formed, mostly per individual. Inter-individual differences in microRNA profiles are bigger than exercise-induced differences. However, several individual microRNAs were significantly regulated either in response to exhaustive exercise, or in response to recovery (Tables C).When allowing unsupervised clustering on expression data of the 30 microRNAs expressed in all samples, it is obvious from the heatmap  and miR-29a-3p (1.3-fold). MiR-29a-3p is the only microRNA that is also regulated after the second exercise session (1.5-fold) (Table 1C), and thus potentially the most reliable biomarker for muscle exhaustion. As shown in Table 2C, multiple microRNAs are regulated in response to a second exercise session. Two microRNAs, miR-99b-5p and miR-151a-3p are downregulated (or depleted) the morning after the first exercise session  (Table 1B), indicating that they may be involved in the recovery process. Unfortunately, these microRNAs did not differ, at any time point, from the PAD samples (Tables D), our pathological model for impaired muscle recovery.Tables D). However, there are several changes in circulating microRNA expression profiles. There are several microRNAs that lose their difference to PAD samples directly after the second exercise session. MiR-193a-5p even loses its significant difference from PAD samples already after the first exercise session. One specific microRNA, miR-495-3p is significantly upregulated in response to both exercise sessions, compared to the PAD samples .As expected, the number of differentially expressed microRNAs between athletes and PAD patients at any given time point is much larger than between time points within the group of athletes (1) via Accession No. GSE117654.The microarray data are available via the Gene Expression Omnibus . Two hours after the time trial however, both microRNAs were significantly downregulated. Both miR-106b-5p and miR-155-5p were also downregulated after the time trial, but both microRNAs normalized rapidly .MiR-29a-3p and miR-193a-5p were both upregulated at 2 h after exhaustive exercise . Both microRNAs appeared upregulated directly after the time trial and decreased in expression during recovery.Of the ‘myomiRs,’ only miR-133b-3p was regulated significantly in response to the time trial, although a similar trend was observed for miR-1-3p (ANOVA p = 0.053) in the TT60 cohort than in the PAD cohort in the later stages of recovery, at 5 h after the time trial.When compared to the PAD cohort, we again observed that microRNA expression profiles differ strongly between elite athletes and patients with critical limb ischemia. Most of the microRNAs that we measured, including miR-193a-5p, miR155-5p, miR133a-3p, miR-133b-3p, and miR-122-5p were lower in athletes than in PAD patients at all time points, both before and after exercise. Consistent with the EEE cohort however, miR29a-3p and miR-495-3p only became different from the PAD cohort after exercise. MiR-29a-3p was significantly lower in the TT60 cohort than in the PAD cohort, directly and at 2 h after the time trial. MiR-495-3p was borderline-significantly lower .All seven snoRNAs could indeed be detected in human plasma. However, especially in the exercise cohort, some snoRNAs were expressed below detection levels in some individual athletes. One snoRNA, SNORD114.1 was significantly upregulated over time after the time trial. SNORD114.1 levels increased gradually throughout the recovery period  or at none or the TT60 time points . Expression levels of the snoRNAs are presented in In this study, we explored the suitability of circulating small non-coding RNAs, including microRNA and snoRNAs, as biomarkers for exercise and muscle damage. We included two cohorts of elite cyclists, subjected to two different types of exercise regimes, as well as a cohort of patients with PAD that were scheduled to undergo lower limb amputation, due to critical limb ischemia. We identified several microRNAs, as well as one snoRNA, that were regulated in plasma in response to strenuous endurance exercise. Furthermore, we identified microRNAs that may indicate impaired suboptimal muscle repair, as they were only differentially regulated from PAD patients, during the recovery phase in elite cyclists.The first thing we noticed, when looking at the microRNA profiling data after repeated exhaustive endurance exercise, was the fact that differences in microRNA expression profiles are much bigger between individuals than between the different time points before, during, and after the 2-day exercise regime. Although we did identify several microRNAs that were significantly regulated in response to exercise, our group of athletes was quite homogenous, consisting only of male elite cyclists in their early twenties. MicroRNA expression is highly variable between individuals and is often age-dependent. Therefore, future replication studies in larger and more heterogeneous cohorts are necessary to determine the most reliable microRNA-biomarkers.We did identify two microRNAs that were significantly regulated in response to the first exhaustive exercise session, miR-29a-3p and miR-193a-5p. MiR-29a-3p was the most consistently regulated, as it was also upregulated on the second day. MiR-29a-3p had also previously been shown to be regulated in response to exercise . Both miMiR29a-3p, like the other microRNAs of the miR-29-family, plays a crucial role in the formation and maintenance of the extracellular matrix and controls, amongst others, the production of collagens. Less is known about the function of miR-193a, but a recent study has shown that miR-193a interacts with High mobility group box-1 (HMGB1) and has effects on angio- and vasculogenesis . HMGB1 iA third microRNA, miR-106b-5p could potentially provide information about the recovery after exercise. MiR-106b-5p is downregulated in response to both exhaustive and endurance exercise, but only immediately after exercise completion (after the second exercise session) and to the 60 min time trial. However, 2 h after the first exhaustive exercise session, and also 2 h after completion of the 60 min time trial, miR-106b-5p have already normalized to the same levels as before exercise. MiR-106b-5p has previously been shown to be highly expressed in skeletal muscle of diabetic patients  and mousFigure 3), we observed that microRNAs are clustered by individual, rather than by time point.When looking at the more classic myomiRs, only miR-133b-3p and possibly miR-1-3p were regulated in response to exercise after the 60 min time trial. But then again, reproducibility in exercise biomarker studies appears relatively low throughout literature. Many different microRNAs, as well as many different cytokines have been reported to associate with various forms of exercise over the years . One expA major finding in the present study was that exercise influences plasma snoRNA levels. snoRNAs have previously been described to be stable in the circulation , howeverFinally, we identified two microRNAs that may function as markers of impaired muscle repair and recovery. Both miR-29a-3p and miR-495-3p are significantly different in athletes compared to PAD patients, but only following exercise. The fact that these microRNAs differ significantly from PAD patients specifically during the recovery stage indicates that plasma levels of these microRNAs may be a marker for muscle repair. Especially miR-495-3p is a promising candidate to mark impaired muscle repair and end-stage PAD, as we have previously shown that miR-495-3p is highly functional in a mouse model for severe PAD . InhibitOur study design has several limitations. First, we did not include non-exercise control groups in our exercise cohorts. Of course, we measured levels of microRNAs and snoRNAs in all participants before starting the different exercise regimes, but as samples were taken at different time point over 2 days, we could not correct for potential confounding factors like circadian rhythm. Similarly, due to our study design where only PAD patients scheduled for limb amputation surgery were included in the study, we did not include samples from PAD patients at other time points. Finally, due to sample depletion, we were unable to confirm the findings from the microarray analyses by qPCR in all the samples of the EEE cohort.In this study, we have investigated the regulation of circulating small non-coding RNAs in response to exercise. MicroRNAs miR-29a-3p, miR-193a-5p, and miR-106b-5p have shown to be significantly regulated in the circulation in response to endurance exercise. MiR-29a-3p and miR-193a-5p may even be able to distinguish between repeated exhaustive and non-exhaustive exercise. MicroRNAs miR-29a-3p and miR-495-3p may mark impaired muscle recovery in patients with severe critical limb ischemia. Furthermore, we showed for the first time that a circulating snoRNA, SNORD114.1, is regulated in response to endurance exercise.KH performed the experiments, analyzed the data, and wrote the manuscript. OS performed the experiments, collected the samples, and edited the manuscript. KS collected the Ampubase biobank samples and edited the manuscript. PQ supervised the Ampubase biobank samples collection and edited the manuscript. JJ designed the protocols, supervised the experiments, and edited the manuscript. AN designed the protocols, supervised the experiments, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Postoperative cognitive dysfunction (POCD) affects a large number of post-surgery patients, especially for the elderly. However, the etiology of this neurocognitive disorder is largely unknown. Even if several studies have reported a small number of miRNAs as the essential modulatory factors in POCD, these findings are still rather limited. The aim of current study was to screen the POCD-related miRNAs in the hippocampus tissues and investigate the target genes of differentially expressed miRNAs and their biological functions underlying POCD pathophysiology.n = 24). The target genes of miRNAs were predicted using the miRWalk3.0 software. Furthermore, we used the ClueGO software to decipher the pathways network and reveal the biological functions of target genes of miRNAs.The miRNA microarray was used to find the abnormal expression of miRNAs in the hippocampus tissues from the POCD model mice to normal mice . The nominal significant results were validated in an independent sample of hippocampus tissues of 10 mice based on same miRNA microarray . Expression level of the most significantly abnormal miRNA was further validated by real-time quantitative polymerase chain reaction (PCR). To determine the expression pattern among miRNAs and genes and detect the interactions, we conducted a weighted gene co-expression network analysis (WGCNA) in the miRNAs and genes expression data from hippocampus tissue of wild type mice  had putative binding sites at 3′-UTR of mmu-miR-190a-3p. Based on protein-protein network analysis, we detected that Tiam1 was a central gene regulated by the mmu-miR-190a-3p.We found that nine miRNAs showed significant associations with POCD in both datasets. Among these miRNAs, mmu-miR-190a-3p was the most significant one. By performing WGCNA analysis, we found 25 co-expression modules, of which mmu-miR-190a-3p was significantly anti-correlated with red module. Moreover, in the red module, 314 genes were significantly enriched in four pathways such as axon guidance and calcium signaling pathway, which are well-documented to be associated with psychiatric disorders and brain development. Also, 169 of the 314 genes were highly correlated with mmu-miR-190a-3p, and four genes  contains supplementary material, which is available to authorized users. Postoperative cognitive dysfunction (POCD), a commonly-seen postoperative complication especially for elderly patients age > 65-year-old), is characterized by cognitive impairments in patients underwent major surgery, and associated with high morbidity and mortality . It cove-year-oldMicroRNAs (miRNAs), which are extensively distributed in eukaryotes, regulate expression of genes by 1) combining with 3-UTR of target gene to inhibit the translation or to degrade target mRNAs at the post-transcription level; and 2) interacting with other target non-coding RNAs, e.g. long non-coding RNA (lncRNA) or circular RNA (circRNA) , 8. A grHence, in this study, by focusing on the biology of the dysregulated miRNAs in POCD, we conducted a comprehensive analysis of miRNAs expression profiles in hippocampus tissue of POCD model mice, and made further investigation of the miRNAs’ potential interaction mechanisms with target genes. Our findings not only extend the understanding of miRNAs’ role in the pathogenesis of POCD but also facilitate the identification of the miRNA biomarkers for improving the prognosis of POCD patients.n = 22, aged 18 months, weights from 40.2 g to 46.6 g) were purchased from SiBeiFu Experimental Animal Science and Technology Co. Ltd.  2016–0002). The mice were individually housed in an air-conditioned room with a temperature of 24 ± 4 °C and 55–65% humidity, under a standard 12 h–12 h light-dark cycle (lights on 6 AM to 6 PM), and had free access to standard food and water. The mice were acclimatized for 1 week before the experiment. The protocol of animal experiment was approved by the Animal Care Committee of the Chinese People’s Liberation Army General Hospital .Healthy aged male C57BL/6 mice  and the anesthetic used was isoflurane. The concentration of the vaporizer was set at 3% and the oxygen flow rate was set at 3 L/min during anesthesia. All mice were deeply anesthetized based on following signs: the slowed rising and falling of chest, no respond to toe pinch, and corneal reflex disappeared. Then, the mice were decapitated by using a guillotine in a uniformly instantaneous manner. The brain was instantly dissected on ice, and the hippocampal tissue was obtained and put into liquid nitrogen. All the mice were decapitated 24 h after operation. The detailed time line of the euthanization of study animals is shown in the Additional file Mice were numbered by weight and randomly divided into two groups: Surgery and Sham, with 11 mice per group. The mice in the Surgery group were exposed to abdominal surgery under local bupivacaine anesthesia , whereaThe MWM test, as a hippocampal-dependent test, was applied to evaluate the spatial learning, spatial memory and cognitive flexibility for the mice . The cogDuring the experiment, mice were released into the water facing the wall of the tank from one of the four quadrants. The mice were trained to find the hidden platform and climb onto it within 60 s. The animals were allowed to stay on the platform for at least 10 s after each trial. If the mice were unable to find the platform in 60 s, it was then placed on the platform for 10 s. After that, the mice were put back to the cage and the second mouse was tested on trial 1. This rotation was repeated until all animals completed trial 1. Subsequently, the process was repeated for subsequent trials until 4 trials completed per day for 5 consecutive days. At 6th day, the platform was removed, and the mice were sent to evaluate their reference memory by being released from the third quadrant. The swimming speed, platform-site crossing numbers, dwelling time in the target quadrant and the escape latency were recorded.n = 3 from each group) by the miRcutemiRNA kit . The nanodrop was utilized to determine the miRNA concentration of each sample according to the optical absorption at 260 nm and the gel electrophoresis was used to detect the miRNA integrity.Total miRNAs were extracted from the hippocampal tissue  was conducted by PREMEDICAL Co. Ltd. . The microarray was utilized to find the aberrant expression of miRNAs from the POCD model mice to normal mice. Fluorescent signals were transformed from picture signal to digital data based on the degree of fluorescent for each probe, and then the data were saved as. DAT files by AGCC software (Affymetrix Genechip Command Console Software). The differential expression analyses were performed by using Transcriptome Analysis Console (v 4.0) and using an FDR correction for multiple testing. Considering that none of the identified miRNAs reached the threshold of adjusted significance, we chose the threshold of a nominal https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE95070), which were deposited by Wei et al. [The miRNA expression profile data of replication cohort were downloaded from the Gene Expression Omnibus (GEO) database  based on the manufacturers’ protocol. Real-time PCR was performed with SYBR Premix Ex Taq kit (Takara Code: DRR041A). The miRNA expression level was evaluated relative to the expression of U6 of the 2 P value of < 0.05 was considered statistically significant.Data were analyzed using GraphPad PRISM . Measurements of dwelling time, number of grossing, escape latency, and speed in MWM test among preoperative and postoperative mice were analyzed by using Student’s t-test. qRT-PCR data were also analyzed with Student’s t-test. A https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE73507). The GSE73507 dataset was designed to gain insight into the relationship between the CAG repeat length and the Huntington disease [n = 24). Because of recent progress of alignment and mapping approaches, which are capable of detecting the transcriptome profiles in a more accurate and effective way compared with former tools [The genes and miRNAs expression data of GSE73507 were acquired from GEO database . The visual gene-gene network plot was displayed by using the Cytoscape version 3.5.1 (https://www.cytoscape.org/) [The R package of WGCNA was used to construct the network modules of highly correlated transcripts subsets . This appe.org/) .Concordance between the highly correlated mRNAs with mmu-miR-190a-3p and those not involved in the module genes were assayed by performing density plots. We compared the distribution of Pearson correlation coefficients of the 169 potential interaction targets of mmu-miR-190a-3p to a control distribution of non-predicted targets which consisted of all other mRNAs that we mapped. The mRNAs which were possibly modulated by mmu-miR-190a-3p displayed more significant negative correlation compared with the control.ClueGO (v. 2.3.4), a plug-in software of Cytoscape, was used to decipher the pathways network and determine their biological functions for the candidate genes . The pothttp://mirwalk.umm.uni-heidelberg.de/) to predict the target genes regulated by mmu-miR-190a-3p [https://string-db.org/cgi/input.pl). By combining the gene-gene interaction result, prediction targets and the protein-protein interaction analysis, we attempted to find the high degree receivable genes regulated by mmu-miR-190a-3p.We conducted further analysis to screen the most potential regulated genes by mmu-miR-190a-3p. We first employed the miRWalk3.0 (-190a-3p . By usinp = 0.002) and the number of crossings (p = 0.049) of mice in Surgery group were significantly decreased than those in Sham group (Table p = 0.660), however, the escape latency increased significantly in the Surgery group (p < 0.001). These results demonstrated that the lower ability to find the platform was not a result from the motor ability, but derived from the spatial learning memory impairment and the cognitive impairment. This indicates that the spatial learning ability of the mice in the Surgery group was impaired notably and a POCD mouse model has been developed.Our MWM test showed that the dwelling time (up Table . The swip < 0.05)  . In this miRNAs expression profile, we detected 85 significantly up-regulation miRNAs and 103 significantly down-regulation miRNAs  = 1.9, p = 2.4 × 10− 3). Real-time quantitative PCR was performed to technically validate the expression pattern of this miRNA in our discovery sample. Consistently, the result showed that the mmu-miR-190a-3p was significantly increased in the hippocampus tissue of mice with POCD (log2(FC) = 1.8, p = 0.041; see Additional file Among of these replicated targets, mmu-miR-190a-3p was the most significantly one in the Surgery group  compared to the mice of the Sham group. For mmu-miR-190a-3p, it was significantly increased in the mice with POCD (Discovery sample: log2(FC) = 2.4, To determine the relevant POCD pathways modulated by target miRNAs, we investigated the orchestrating of the transcriptome in the hippocampus of mice brain by performing weighted gene co-expression network analysis to the 24 specimens as described in Materials and Methods. We detected 25 modules with highly significant co-expression patterns , and the second to fourth pathways were calcium signaling pathway (p = 7.7 × 10− 08), Fc gamma R-mediated phagocytosis (p = 1.2 × 10− 2), and Fat digestion and absorption (p = 5.0 × 10− 02), respectively. Among these four significant pathways, the axon guidance, calcium signaling pathway and Fc gamma R-mediated phagocytosis have been widely identified to be related with the psychiatric disorders and brain development [A total of 314 genes and 5 miRNAs were grouped in the red module  involved putative binding sites at 3′-UTR and CDS regions of mmu-miR-190a-3p , Tiam1 and Ntrk3 , respectively. As for Arhgef25, we observed a marginally significant correlation with the mmu-miR-190a-3p . Importantly, as showed in Fig. Tiam1 as a hub gene was possibly dysregulated by mmu-miR-190a-3p.As a result, we found that 4 of 169 correlated genes  required high level expression in development of spine and synapse, might be repressed by mmu-miR-190a-3p in the POCD patients.In this study, we examined dysregulated miRNAs in the hippocampus of POCD mice and further analyzed the potential mechanisms of the target miRNAs in this neurocognitive disorder. Firstly, based on two independent miRNA microarray datasets, we identified 9 significant dysregulated miRNAs in the hippocampus tissues of POCD mice. Among of these identified miRNAs, mmu-miR-190a-3p was the most significantly differential expressed one in both datasets, and this had been validated by using the qRT-PCR. Secondly, we used a sequence-based dataset of both mRNA and miRNA data to perform co-expression analysis, which revealed 25 co-expression modules. Interestingly, the co-expression red module including mmu-miR-190a-3p was of significant enrichment in the neuron-related pathways and immunology pathways, which have been well-documented to play crucial roles in the psychiatry diseases –38. FinaP = 0.054). In addition, Shan et al. illuminated that the expression of mmu-miR-30a was significantly decreased in the mPFC and hippocampus of aged rats compared with control and was involved in the expression regulation of several innate immune system related genes [P = 0.046).Previous studies have reported that several miRNAs were significantly associated with POCD , 18–20, ed genes . Such a Tiam1 and Arhgef25, were involved in the interaction network. Interestingly, Tiam1, as a hub gene located on the central of network map, might be modified by abnormal expression of mmu-miR-190a-3p and played important roles in the pathogenesis of POCD.Furthermore, we also performed an integrative analysis to refine a collection of putative mRNA-miRNA interaction pairs for mmu-miR-190a-3p. By applying to a powerful approach, which was combining expression data of miRNAs and mRNAs while performing the co-expression analysis based on Pearson correlation, we provided a new clue to detect the biological network relationship between miRNA and mRNA –44. BecaTiam1 is widely expressed in the developing central nervous system [Tiam1 is one of the important Rac-specific GEFs which coordinate activation of the GTPase Rac1 with other GEFs in many partitioning-defective (PAR) processes within spines [Tiam1 activates and recruits to the membranes after exposure to amyloid beta peptide in hippocampal neurons, and this might exert influence to the pathology of Alzheimer disease [Tiam1 knockdown declined hippocampal neuronal vulnerability to oxygen/glucose depravation [Tiam1 is an important gene for the hippocampal neurons’ development, and for the etiology of several psychiatry diseases. We showed here that increasing expression levels of mmu-miR-190a-3p might inhibit the activation of the Tiam1/Rac1 pathway in the hippocampus after surgery.The majority of excitatory synapses existed in micron-sized dendritic protrusions of the mammalian brain, which can change along with the variation of synaptic activity in terms of the alternation in number and shape . Tiam1 is system . Tiam1 in spines . The PARn spines , 49. Thin spines –52. Par3n spines . Due then spines , 54. If  disease , 56. A sravation . ConclusTrim1gene which may be restrained by mmu-miR-190a-3p in the hippocampus of POCD, we did not confirm these gene-miRNA interactions experimentally, which should be the research focus of future study.There were a few of limitations in this study. First, due to the limited number of sample size and low expression level of miRNA in brain, some weak signals of miRNAs were difficulty to be observed. To obtain more reliable expression signals, we used both the public data from GEO database and RT-PCR to validate the significantly differential expressed miRNAs. Second, although we employed various robust bioinformatics methods to find the binding targets of mmu-miR-190a-3p, such as the expression of Tiam1 was inhibited by mmu-miR-190a-3p in the hippocampus tissue of POCD model mice. These results demonstrated that mmu-miR190a-3p probably serve as an important regulator to inhibit target genes expression in the hippocampus tissue of POCD patients. More molecular-based studies are warranted to explore the underlying biological mechanisms of target genes by mmu-miR-190a-3p in the pathogenesis of POCD.To sum up, we provided robust evidence supporting that mmu-miR-190a-3p has a strong positive correlation with the incidence of POCD. The pathway analysis displayed that these mmu-miR-190a-3p targeted genes were enriched in the psychiatry-related pathways and immune system-related pathways. Among of these target genes, Additional file 1:Table S1. The primer sequence of the validated miRNA. i.e., the primer sequence of mmu-miR-190a-3p used for real-time PCR assay. Table S2. Predictionbinding sites for mmu-miR-190a-3p. The data were predicted by using miRwalk 3.0 for obtaining reliable targets for mmu-miR-190a-3p. Table S3. Genes and miRNAs involved in red module. This red module were generated by using our co-expression weighted network analysis by combined the 314 mRNA and 5 miRNAs data. (DOCX 49 kb)Additional file 2:Figure S1. The detailed time line of the euthanization of study animals. According to the protocol, all mice were euthanized before obtaining hippocampal tissue. All mice were deeply anesthetized based on several signs  was downloaded from the GEO database. Figure S3. The results of qRT-PCR for technical replication. Expression level of the most significantly mmu-miR-190a-3p was validated by using real-time PCR assay. Reverse transcription reaction was performed with M-MLV Reverse Transcriptase kit (Takara Code: D2639A) based on the manufacturers’ protocol. Figure S4. WGCNA module-based analysis for genes and miRNAs expression data. The genes and miRNAs expression data of GSE73507 were acquired from GEO database. After the filtering process, 13,241 mRNAs and 546 miRNAs from hippocampus tissue of wild type mice (n = 24) were included for WGCNA analysis. Figure S5. KEGG pathway analyses for mmu-miR-190a-3p highly related genes in red module. We refined 169 genes from red module with a highly connected with mmu-miR-190a-3p (Fig. igns see  part. Th


The monolithic integration of an ultraviolet (UV) sensor and warning lamp would reduce the cost, volume, and footprint, in comparison to a hybrid combination of discrete components. We constructed a module comprising a monolithic sensor indicator device based on basic p–i–n (PIN) photodiodes and a transimpedance amplifier. GaN-based light-emitting diodes (LEDs) with an indium-tin oxide (ITO) current-spreading layer and PIN photodiodes without ITO deposition on the light-receiving area, were simultaneously fabricated. The resultant incident photon-to-electron conversion efficiencies of the PIN photodiodes at UV wavelengths were significantly higher than those of the reverse-biased LEDs. The photocurrent signals of the PIN photodiode were then converted to voltage signals to drive an integrated visible LED, which functioned as an indicator. The more the ambient UV-light intensity exceeded a specified level, the brighter the glow of the LED. The responsivities of 0.20 and 0.16 A/W were obtained at 381 and 350 nm, respectively, under a bias voltage of 5 V. We also addressed the epitaxial structural details that can affect the collection efficiency of the photocurrent generated by UV light absorption. The crosstalk between the PIN photodiode and LEDs (of various center-to-center distances) was measured. Ultraviolet (UV) light detection has become increasingly important. In particular, UV light-emitting diodes (LEDs) have been applied in various fields, such as epoxy curing, air and water sterilization, and surface disinfection. Appropriate UV light monitoring is essential to protect people from UV damage. UV sensors can also be used in flame detection, biomedical spectroscopy, and military applications ,2. III-n0.2Ga0.8N layer (doping concentration = 3 × 1017 cm−3), 15-pair In0.12Ga0.88N/GaN multiple quantum wells , a 100-nm n-GaN layer, and a few-micron n+-GaN layer. For the efficient collection of photocurrent, the p-electrode of the photodiode was located at the center of the device. The processing procedure is described as follows. Each device was isolated by a deep etch that extended down to the insulating sapphire substrate through inductively coupled plasma reactive-ion etching (ICP-RIE). Next, ICP-RIE was used to produce mesas on the LED and photodiode, which provided access to the n-GaN layer. Subsequently, a SiO2 insulation/passivation layer was deposited. Next, an electron beam evaporator was used to deposit an ITO layer with a thickness of approximately 200 nm and a composition of 5 wt% SnO2 and 95 wt% In2O3. The ITO layer was then thermally annealed at 625 °C in ambient N2 for 8 min; the two functions of the ITO were current spreading for LEDs and ohmic p-contact for both devices. Finally, p/n-metal electrodes were formed. The device structure of the PIN photodiode and LED is illustrated in 2, which is equivalent to the mesa area of 360 μm × 360 μm subtracted by the size of the p-metal pad, that is, 100 μm × 100 μm. Compared with the LED, the photodiode had a significantly lower slope efficiency—which is defined as the quotient of the optical output power and injection current—in addition to a marginally higher forward voltage and nearly equal series resistance. These photodiode characteristics are attributable to the considerably smaller ITO layer on the photodiode than on the LED. This smaller ITO layer decreased current-spreading area, which resulted in a decreased light-emitting area and marginally increased total resistance. Moreover, the external quantum efficiency (EQE) of the photodiode and reverse-biased LED were measured by employing an incident photon-to-electron conversion efficiency (IPCE) apparatus, which comprised a broadband 300-W Xenon lamp , monochromator , chopper, and lock-in amplifier . The results are plotted in R at low frequencies. The circuit bandwidth in this design was approximately 1.6 kHz, which was considerably larger than the current operating frequency of approximately 5 Hz. Specifically, we operated the module at such a low frequency that we could visually inspect the on-off switch of the LED despite the persistence of vision. The response speed of a PIN photodiode is of the order of nanoseconds [R) value was configured to be such that the LED indicator was switched on once a specified UV light intensity was exceeded. Assuming that the specified UV intensity level was 1.5 mW/cm2 at 389 nm, the associated illuminating current at a UV intensity of 1.7 mW/cm2 should, in theory, be converted to an output voltage (V1) larger than the turn-on voltage of the LED. We built a sensor indicator module that detects UV light through a PIN photodiode. Specifically, the photodiode converts photocurrent signals to voltage signals through an external transimpedance amplifier, and the resulting voltage signals are used to drive an integrated LED as the indicator. The schematic of the designed module is displayed in oseconds . The resA GaN-based UV sensor and indicator module comprising an integrated PIN photodiode and LED was designed and tested. For UV detection, photocurrent signals from UV light were converted to voltage signals to drive an integrated LED (functioning as a warning lamp). The output power can be scaled up by using multiple or large LEDs when crosstalk is contained. Responsivity in the UV range was improved by (1) having a thin GaN cap layer, (2) having low doping in the p-cladding layer, and (3) not having ITO deposition on the light-receiving area. The responsivities of 0.20 and 0.16 A/W were obtained at 381 and 350 nm, respectively, under a bias voltage of 5 V. The designed device could be valuable to LED manufacturers.


The morphology and microstructure of the HBP-NH2-grafted bagasse cellulose (HBP-g-BC) were characterized and its adsorption capacity for Cr(VI) ions in aqueous solutions was investigated. The rough surface structure of HBP-g-BC that is beneficial for improving the adsorption capacity was observed by scanning electron microscopy (SEM). The grafting reaction was confirmed by Fourier-transform infrared (FT-IR) spectroscopy. The adsorbent performance was shown to be better with a lower pH value, a higher adsorbent dosage, or a higher initial Cr(VI) concentration. Moreover, the kinetics study revealed that the adsorption behavior followed a pseudo-second-order model. The isotherm results showed that the adsorption data could be well-fitted by the Langmuir, Freundlich, or Temkin models. Moreover, HBP-g-BC could maintain 74.4% of the initial removal rate even after five cycles of regeneration. Thus, the high potential of HBP-g-BC as a bio-adsorbent for heavy metal removal has been demonstrated.A novel bio-adsorbent was fabricated via grafting an amino-terminated hyperbranched polymer (HBP-NH Chromium is one of the most common heavy metals used in industry and has a broad range of industrial applications, such as electroplating and metal processing. It is well-known that chromium ions Cr(VI) possess significantly higher toxicity than other forms of chromium. Without proper treatment, wastewater containing Cr(VI) may cause serious environmental problems ,2. WasteFor the removal of heavy metals from wastewater, different methods, such as precipitation, solvent extraction, ion exchange, membrane filtration, and adsorption ,6,7, havThe adsorption process uses adsorbents to remove heavy metals. During the adsorption, heavy metals are transferred from wastewater and adhered to the surface of the adsorbent by the physical and/or chemical interactions between them. Among various water treatment processes, adsorption is considered to be one of the most efficient techniques because of its good operational flexibility, high efficiency, and excellent reusability ,14. In rCellulose is a natural biopolymer that is widely available and environmentally friendly. It has high potential to be developed into bio-adsorbents for wastewater treatment ,19. As cSugarcane bagasse is an important by-product of the sugarcane industry. There has been a growing interest in finding new applications for this cheap resource. Although the adsorption capacity of coarse sugarcane is not satisfactory, chemically modified bagasse cellulosic materials have shown great potential to be used as effective adsorbents for heavy metals ,27,28. YHyperbranched polymers (HBPs) are a type of macromolecule with highly branched three-dimensional (3D) structures. Compared with their linear and crosslinked analogs, HBPs exhibit properties such as lower viscosity, higher solubility, and less entanglement due to their unique globular and dendritic architectures. They have been widely used in fields such as coating, nanotechnology, additives, and biomaterials . Since H2) and evaluating the adsorption capacity of this bio-adsorbent for Cr(VI) ions in aqueous solutions. The morphology and microstructure of the fabricated HBP-NH2-grafted bagasse cellulose (HBP-g-BC) were characterized by SEM and FT-IR. The effects of pH, adsorbent dosage, and initial Cr(VI) concentration on the adsorption behavior of the bio-adsorbent were also examined. Moreover, the adsorption mechanism was explored by a kinetics and an isotherm analysis.This work aimed at fabricating a novel bio-adsorbent based on sugarcane bagasse cellulose (BC) grafted with an amino-terminated hyperbranched polymer . Anhydrous sodium sulfate (AR), sodium periodate (AR), and ethylene glycol (AR) were purchased from Kelong Chemical Reagent Factory . Diethylenetriamine (AR), methyl acrylate (AR), methanol (AR), glacial acetic acid (AR), and anhydrous ethanol (AR) were purchased from Sinopharm Chemical Reagent Co., Ltd. . Potassium dichromate (AR) was supplied by Guanghua Chemical Reagent Factory . Deionized water was made in the lab and used in all the experiments.Dialdehyde bagasse cellulose (DABC) was prepared according to Huang et al. . Coarse 2 was prepared by the polycondensation procedure introduced by Wang et al. [2 and AB3 monomers were obtained. These two types of monomers were transferred to an eggplant flask attached to a rotary evaporator to react. During the reaction, firstly the methanol was removed by decompression. Then, the temperature was increased to 150 °C and the reaction was carried on under decompression for another 4 h to obtain HBP-NH2.HBP-NHg et al. . An amou2 methanol solution (the volume ratio of HBP-NH2 to methanol was 1:1) was added to react with DABC for 7 h to obtain HBP-g-BC. Afterwards, the obtained cellulose was washed with deionized water and ethanol and was then filtered by suction to form a filter cake. The final product was obtained by vacuum-drying the filter cake under 40 °C until it reached a constant weight.Ten grams (10 g) of dried DABC powders and 100 mL of methanol were added to a three-necked flask. They were mixed by stirring and heating at 45 °C. When the temperature of the solution reached 45 °C, 5 mL of glacial acetic acid was added for another 20–30 min of reaction. Then, 40 g of an HBP-NHg-BC were measured by a Fourier-transform infrared spectrometer . A small amount of sample was mixed with KBr powder. Then, the blend was ground and compressed to form the testing disc. The spectral range of 4000–400 cm−1 was used for analysis of each sample.The Fourier-transform infrared (FT-IR) spectra of BC, DABC, and HBP-g-BC were observed by a field emission scanning electron microscope  with an accelerating voltage ranging from 0.02 to 30 kV and a resolution of 0.8 nm.The surface morphologies of BC and HBP-g-BC were measured by a simultaneous thermal analyzer . The samples were heated from 35 to 900 °C with a heating rate of 10 °C/min.The thermal stabilities of BC and HBP-Solutions were prepared to simulate wastewater produced from a metal processing unit . A certag-BC was added to the prepared Cr(VI) solution at a certain temperature and a pH value. The adsorption was carried out in a rotary shaker at a speed of 200 rpm/min for different time periods.To conduct a batch mode adsorption test, a certain amount of HBP-g-BC. For each set of conditions, the measurements were carried out in triplicate for different samples to ensure the reproducibility of the data. The mean values were calculated and used for further analysis.A series of batch adsorption tests were conducted to investigate the effects of pH, adsorbent dosage and initial Cr(VI) concentration on the adsorption performance of HBP-g-BC was determined by the adsorption amount q and the removal rate R, which can be calculated by the following two equations.The metal ion adsorption capacity of HBP-C0 and eC are the concentrations (mg/L) of metal ions in the solution before and after the adsorption, respectively, V is the volume (L) of the aqueous solution, and M is the mass (g) of HBP-g-BC consumed during the adsorption.In these equations, g-BC was added to 50 mL of Cr(VI) solution (20 mg/L) at 35 °C and pH = 3 in a conical flask. The adsorption process was carried out for 4 h. Afterwards, the adsorbent was treated with 0.1 mol/L HCl for 24 h and was then filtered. The filtrate was used for desorption analysis. Then, the adsorbent was washed with distilled water to remove the residual acid and to ready it for the next desorption test. The above procedure was repeated for five cycles to evaluate the reusability of the adsorbent.For adsorption, 0.1 g of HBP-g-BC are shown in −1 is considered to be due to the hydroxyl (–OH) stretching. The characteristics peak around 2900 cm−1 could be ascribed to the C–H vibration. Compared with that of BC, the spectrum of DABC shows a characteristic adsorption peak of carbonyl group at 1726 cm−1, suggesting that aldehyde groups were introduced to BC to form DABC [−1, and there is a new characteristic peak at 1544 cm−1, which could be assigned to the vibration of amine groups. This indicates that through the reaction between the aldehyde groups of DABC and the amino groups of HBP-NH2, HBP-NH2 was successfully grafted onto BC to form HBP-g-BC [The FT-IR spectra of BC, DABC, and HBP-orm DABC . In the HBP-g-BC .g-BC are presented in g-BC had some small peaks with irregular shapes. This surface feature presenting a higher specific surface area could lead to an increased number of active adsorption sites and thus an improved adsorption capacity of HBP-g-BC.The SEM images of BC, DABC, and HBP-g-BC. Three stages of weight loss can be observed for both BC and HBP-g-BC. At the initial stage (35–242 °C for BC and 35–172 °C for HBP-g-BC), the weight loss of BC and HBP-g-BC was 2.7% and 11.2%, respectively. This first-stage weight loss could be mostly ascribed to the moisture evaporation. The major weight loss occurred at the second stage (242–400 °C for BC and 172–400 °C for HBP-g-BC), during which the weight losses for BC and HBP-g-BC reached 73.6% and 56.9%, respectively. This second-stage weight loss could be attributed to the degradation and decomposition of BC and HBP-g-BC. The onset temperature for the significant weight loss of HBP-g-BC was lower than that for BC. Regarding this, the introduction of HBP-NH2 groups might make the polymer easier to decompose. The results are consistent with those in previous studies, where the decomposition temperature of cellulose usually decreased by grafting with hyperbranched polymers [polymers .g-BC. The protonation of amino groups was more active under acidic conditions. In this case, the surface of HBP-g-BC was strongly positively charged. Moreover, since Cr(VI) mainly exists in the solution in the forms of HCrO4− and CrO42− at low pH values, the strong electrostatic attraction makes it easy for the adsorbent to capture negatively charged Cr(VI) ions, leading to a higher adsorption capacity. On the contrary, high pH values usually reduce the degree of protonation of amino groups, which might negatively influence the adsorption performance of HBP-g-BC.The pH of an aqueous solution significantly affects the ionic forms of heavy metals and the surface charge of an adsorbent in the solution. Therefore, pH strongly influences the adsorption capacity of the adsorbent for Cr(VI) ions. Cr(VI) is more reactive at low pH values because its reduction potential decreases with increasing pH. In our preliminary experiments, the change in adsorption capacity is small when the pH value is higher than 6. To investigate the effect of pH, the adsorption tests were carried out at pH values from 2 to 6 . The relAdsorbent dosage is an important parameter that should be evaluated and controlled in the wastewater treatment. The results  show thag-BC is an excellent adsorbent that can maintain a stable removal rate by increasing its adsorption capacity with increasing initial concentration.The adsorption kinetics measurements were conducted at 25, 35, and 45 °C. To investigate the kinetics of the adsorption process, the pseudo-first-order model (Equation (3)) and the pseudo-second-order model (Equation (4)) were used to evaluate the experimental data . The equeq and tq (mg·g−1) are the amount of the Cr(VI) ions adsorbed at equilibrium and time t (min), respectively; k1 (min−1) is the pseudo-first-order rate constant of the adsorption; and k2 (mg·min−1) is the pseudo-second-order rate constant of the adsorption.In these equations, As shown in g-BC adsorbent, three widely used isotherm models, namely Langmuir (Equation (5)), Freundlich (Equation (6)), and Temkin (Equation (7)), were used to examine the experimental data [The adsorption isotherm model provides a relationship between the amount of adsorbed metal ions and that of the non-adsorbed metal ions in a solution when the equilibrium was reached. It is critical for predicting the adsorption behavior and comparing the adsorbent performance under different conditions . To thortal data :(5)1qe=1eq (mg/g) is the amount of metal ions adsorbed at the equilibrium; eC (mg/L) is the concentration of metal ions in the solution at the equilibrium; LK is the Langmuir constant (L/mg); FK and 1/n are the Freundlich constants related to the capacity and intensity of the adsorption, respectively; TA (L/g) is the Temkin equilibrium constant; Tb is the Temkin constant associated with the adsorption heat; R (8.314 J/mol) is the gas constant; and T (K) is the absolute temperature.In these equations, g-BC, which could easily lead to multilayer physical adsorption on its surface [The isotherm model parameters for Cr(VI) ions under three different temperatures are summarized in  surface . The Lan surface . The Tem surface .RL expressed as RL = 1/(1 + KLC0) was calculated using the Langmuir model. RL can be used to effectively evaluate whether the adsorption isotherm process is favorable (0 < RL < 1) or unfavorable (RL > 1). The RL values for 15, 25, and 35 °C were 0.505, 0.472, and 0.417, respectively, indicating that the adsorption process was favorable. This result corresponds to the n values  calculated by the Freundlich model, since n values in the range of 1 to 10 also suggest a favorable adsorption process. Furthermore, with the increased temperature, both qmax and KL in the Langmuir model were increased. This suggests that the adsorption of Cr(VI) by HBP-g-BC was an endothermic process, which means a higher temperature can effectively accelerate the adsorption process.The constant g-BC might occur in three consecutive stages. Firstly, anionic Cr(VI) ions bind with the positively charged amino groups of HBP-g-BC. Secondly, Cr(VI) reduces to Cr(III) by adjacent electron-donor groups. Finally, Cr(III) reacts with the functional groups on HBP-g-BC to form a Cr(III) complex. It is worth noting that the maximum adsorption capacity of HBP-g-BC for Cr(VI) at 25 °C was 75.36 mg/g. In our preliminary studies, the maximum adsorption capacity of DABC was 11.27 mg/g under the same conditions. It is evident that the grafting of HBP-NH2 significantly improved the adsorption capacity of BC.These results are in agreement with previous research . The posg-BC after up to five consecutive adsorption–desorption cycles are presented in g-BC had good reusability and thus has great potential to be used as an economical adsorbent.The removal rates for Cr(VI) of regenerated HBP-g-BC bio-adsorbent is capable of removing Cr(VI) ions from aqueous solutions, and its maximum adsorption capacity for Cr(VI) was much higher than that of DABC. The introduction of HBP-NH2 groups onto BC could lead to a rough and irregular surface, which is instrumental to Cr(VI) adsorption. We also found that the adsorption performance was highly dependent on the pH, adsorbent dosage, and initial concentration of Cr(VI). A high adsorption capacity could be achieved at a low pH value due to the surface protonation of HBP-g-BC. The removal rate was increased with a higher adsorbent dosage as a higher amount of the adsorbent could provide more active adsorption sites. With the increased initial Cr(VI) concentration, the adsorption capacity was also increased. Regarding this, the stronger driving force at larger concentration gradients could facilitate the contact between Cr(VI) ions and the adsorbent. We found that the adsorption kinetics data can be accurately described by the pseudo-second-order model rather than by the pseudo-first-order model. The adsorption isotherm data are in good agreement with the Freundlich, Langmuir, and Temkin models, which means that the adsorption of Cr(VI) is an endothermic and favorable process. Moreover, HBP-g-BC could maintain 74.4% of the initial removal rate even after five cycles of regeneration. Thus, our work has shown the high potential of HBP-g-BC as a bio-adsorbent for heavy metal removal and wastewater treatment.Here, we have demonstrated that the HBP-